The expanding field of biotechnology provides the tools for scientists to introduce important traits into a variety of plant species. New technologies promote the production of commercially viable transgenic crops and have a significant economic impact on the agricultural industry. These advancements enable creation of new crop germplasm containing desirable novel traits. Such traits include improvements in the nutritional quality, insect resistance, disease resistance, and yield of many crops. Cotton is the leading fiber crop worldwide and holds significant agronomic influence in a number of markets. Accordingly, much effort is continually directed toward the genetic engineering of this agronomically important crop species.
Genetic engineering of plants is essentially a two-step process: transformation and regeneration. First, plant cells are transformed, thereby introducing a nucleic acid sequence that is typically integrated into the genome of the host cell. Second, a sexually competent plant is regenerated from the transformed cells. This regeneration step comprises an induction and a germination phase. The nonembryogenic cotton tissue is induced, under suitable culture conditions, to form embryogenic cotton calli. The embryogenic cotton calli mature, and embryos may then be germinated to form plants. The transformation and regeneration processes preferably are complementary such that the successfully transformed tissues are capable of developing into competent whole plants.
Several methods are available for introducing DNA sequences into plant cells and are well known in the art. Suitable methods include, but are not limited to, bacterial infection, binary bacterial artificial chromosome vectors, direct delivery of DNA (e.g., via PEG-mediated transformation, desiccation/inhibition-mediated DNA uptake, electropbration, agitation with silicon carbide fibers, and acceleration of DNA-coated particles (reviewed in Potrykus, Ann. Rev. Plant Physiol. Plant Mol. Biol., 42: 205, 1991).
Methods for transforming dicots primarily use Agrobacterium tumefaciens. Transgenic plants reported include cotton (U.S. Pat. No. 5,004,863 and U.S. Pat. No. 5,159,135). These patents describe the overall regenerative process comprising transformation and selection of a transformed plant tissue, induction of that tissue to form embryos, and germination of those embryos to form a plant. Various media compositions are reported to promote the process. Embryogenesis reportedly required several months.
U.S. Pat. Nos. 5,244,802, 5,583,036, and 5,695,999 disclose methods for regenerating cotton plants from somatic cells. Modified media compositions were reported to be useful at different stages of the regenerative process. More specifically, transformed plant tissue was grown in media supplemented with glucose until phenolic secretions ceased, whereby the tissue was transferred to a media supplemented with sucrose instead of glucose. Many of the cotton lines tested formed transgenic calli but did not undergo embryogenesis and regenerate into a plant.
U.S. Pat. No. 4,672,035 describes a process of regenerating cotton plants utilizing modifications in media composition. Proembryoids were obtained in 1-6 months. Root initiation and growth were reportedly promoted by lowering the glucose concentration in the media.
There exists a need in the art for improved methods for the transformation and regeneration of cotton plants. Such methods may be useful to promote the engineering of desirable traits into this agronomically important crop.